Drug resistant strains of human immunodeficiency virus type 1 (HIV-1) pose a significant challenge for effective long-term treatment of infection. One counter-measure against resistant strains of virus is treatment with therapeutics targeted to novel areas of HIV-1 replication. The long term goal of our research is to elucidate the assembly and transport of the HIV-1 preintegration complex (PIC) to aid in the development of novel antiretroviral therapeutics. The PIC facilitates the infection of cells with HIV-1 by targeting the viral DNA into the nuclei of cells after reverse transcription. Due to challenges in producing and purifying sufficient quantities of PICs there is limited understanding of their assembly, composition, and mechanism of nuclear import. Consequently, there are neither existing inhibitors of PICs nor any in development. The objective of this proposal is to identify and characterize novel cellular components of HIV-1 PICs. We have developed two complementary methods to reproducibly produce sufficient quantities of functional PICs for analysis by mass spectrometry. The feasibility of these approaches is demonstrated by the successful detection of known HIV-1 PIC components and the identification of several candidate proteins. At least one candidate thus far, LRP130, has been found to be critical for HIV-1 infection. These proposed studies will continue the investigation of role of LRP130 and other candidate proteins in HIV-1 replication using gain-of-function and loss-of-function assays. We will also identify additional PIC-associated cellular proteins using two innovative strategies. First, we will perform quantitative 2-D-DiGE proteomic analysis on PICs purified by velocity gradient centrifugation. Second, we will affinity purify HIV-1 protein complexes using a novel in vivo biotinylation method. Successful completion of these studies will identify new cellular co-factors critical for the early steps of HIV-1 replication, advance the understanding of PIC assembly and transport, and discover new targets for the development of antiviral therapies. The Specific Aims are: 1. To determine the role of identified candidate proteins in HIV-1 replication. 2. To identify additional cellular proteins associated with HIV-1 PICs during early replication. 3. To characterize new candidate proteins.